The present invention relates generally to optical microscopy systems and methods, and more particularly to systems and methods for producing circular extinction (CE) contrast images, including circular dichroism (CD) images.
Circular dichroism (CD) is the differential absorption (circular extinction, CE) of left- and right-circularly polarized light (CPL) traversing a sample. CD reveals the dis-symmetry of a molecule's chromophores which then also exhibit circular birefringence (CB), manifested in optical rotation (OR), i.e., the change in the azimuth of light passing through chiral substances that results from different refractive indices for left and right CPL.
The idea of a CD microscope for anisotropic samples is not new. In 1982, Maestre and Katz adapted a Carey spectropolarimeter to a microscope for single point measurements of the CD spectra of chromatin. (See, Maestre, M. F.; Katz, J. E. Biopolymers, 1982, 21, 1899–1908. See also: Maestre, M. F.; Salzman, G. C.; Tobey, R. A.; Bustamante, C. Biochemistry, 1985, 24, 5152–5157; Livolant, F.; Mickols, W.; Maestre, M. F. Biopolymers, 1988, 27, 1761–1769; Livolant, F.; Maestre, M. F. Biochemistry, 1988, 27, 3056–3068). They faced instrumental artifacts (see, Shindo, Y.; Nishio, M.; Maeda, S. Biopolymers, 1990, 30, 405–413; Sindo, Y.; Ohmi, Y. J. Am. Chem. Soc. 1985,107, 91–97) arising from electronic polarization modulators in commercial instruments that typically generate sinusoidally varying polarization states, (see, Hipps, K. W.; Crosby, G. A. J. Phys. Chem. 1979, 83, 555–562) thereby introducing a small admixture of linearly polarized light into the circularly polarized output. Residual ellipticity, when coupled with the LB and LD of ordered media, generates artifactual CD signals. (See, Schellman, J.; Jensen, H. P. Chem. Rev, 1987, 87, 1359–1399 and Disch, R. L; Sverdlik, D. I. Anal. Chem. 1969, 41, 82–86). Strain in photoelastic modulators (PEMs) compounds these artifacts. (See, Nordén, B. Acta Chem. Scand. 1972, 26, 1763–1776; Davidsson, Å, Nordén, B. Spectrochim. Acta, Pt. A, 1976, 32, 717–722; Davidsson, Å; Nordén, B.; Seth, S. Chem. Phys. Lett. 1980, 70, 313–316) Attempts have been made to skirt these problems by adding additional modulators, (see, Cheng, J. T.; Nafie, L. A.; Stephens, P. J. J. Opt. Soc. Am., 1987, 65, 1031–1035) rotating the sample, (see, Tunis-Schneider, M. J. B.; Maestre, M. F., J. Mol. Biol. 1970, 52, 521–541; Nuckolls, C.; Katz, T. J.; Verbiest, T.; Van Elshocht, S.; Kuball, H. G.; Kiesewalter, S.; Lovinger, A. J.; Persoons, A. J. J. Am. Chem. Soc. 1998, 120, 8656–8660) and performing complex analytical transformations of independent chiroptical measurements. (See, Kuball, H.-G.; Altschuh, J. Chem. Phys. Lett. 1982, 87, 599–603) Most recently, Kuroda, in collaboration with JASCO, made advances by tailoring a single point CD spectropolarimeter for solid-state samples by selecting a photomultiplier tube with the smallest polarization bias and a PEM with the least residual static birefringence. (See, Kuroda, R.; Harada, T.; Shindo, Y. Rev. Sci. Instr. 2001, 72, 3802–3810).
Despite its widespread use in structure determination, CD and OR spectroscopy is woefully under-utilized, especially in the analysis of organized media that exhibit linear anisotropies.
The phenomenon of refractive index anisotropy and absorption anisotropy is called linear birefringence (LB) and linear dichroism (LD). In isotropic media, (LD) and (LB) disappear enabling the measurement of CD. In practice, this was not routine until the 1960s, when electro-optic circular polarization modulators were incorporated in commercial instruments. With electro-optic modulation, λ/4 retardation may be achieved in a crystalline material through an applied electric field. Photoelastic modulators (PEMs), strain sensitive materials oscillated via the electrostrictive effect, are the current standard.
In anisotropic media, LB and LD obscure OR and CD; the latter are often three or four orders of magnitude smaller. When a sample is sufficiently thin, two orthogonal, linearly-polarized eigenrays emerge as a coherent superposition, now containing the phase difference δ, where =2πΔnL/λ, where L is the thickness and Δn is the difference in the refractive indices (or liner birefringence (LB)). A sample that absorbs one of the orthogonal polarized light beams in preference to the other displays absorption anisotropy, called linear dichroism (LD). Measurements of LB or LD probe macroscopic structure in terms of the alignment and orientation of its components as sampled by the eigenrays.
Measuring OR or CD in organized media may be likened to searching for a needle in a haystack. Ever since OR was discovered in 1811 by Arago, measurements of chiroptical effects have been nearly impossible for anisotropic samples. It is therefore not surprising that the most recent measurements on oriented or solid samples have focused on uniaxial, nematic liquid crystals, films, and powders.
The difficulty of measuring CD of organized media with commercial instruments is so extreme that it is practically assumed from the start that CD measurement is a technique restricted to isotropic solutions. In practice, scientists typically consign CD measurement to unoriented samples and LD measurement to oriented samples, as if the complementary techniques were mutually exclusive.
Part of the problem stems from the fact that electronic modulators typically generate sinusoidally varying polarization states instead of rectangular waveforms, introducing a small admixture of linearly polarized light into their circularly polarized output and thereby preventing the straightforward separation of LB and LD in an anisotropic sample. This will appear as a CD signal in a commercial spectrometer even if the sample's true CD=0. Strain of a photoelastic modulator (PEM) tends to compound these artifacts. Attempts to overcome deficiencies of the above modulation techniques by adding additional modulators, rotating the sample and performing complex analytical transformations of independent chiroptical measurements have been stymied by defects in both the optical train and the phase modulation.
Because CD comes from a small difference in absorbance (as small as 1 part in 104), a reasonable resolution could be achieved in the world before CCD cameras, but only with fast sampling times in electronic modulators (10–100 KHz) and photomultiplier tubes. Why then not use electronic polarization modulation with CCD detection to make images? CCDs operate at about 1 KHz. Being much slower than PEMs they are incompatible with them. While others are trying to force compatibility by speeding up the CCD or slowing down the modulation, these designs remain constrained by limited spectral ranges (<80 nm), noise resulting from the simultaneous operation of two detectors, and sizable deviations from perfect circular polarization (e.g., parasitic ellipticities).
Accordingly, it is desirable to provide a CE contrast imaging system in combination with a CCD detector or other imaging device to provide useful CE contrast images, including CD images. Further, such an imaging system should avoid the use of electric polarization modulation so as to avoid imperfections in circular polarization and thereby improve the quality of CD images.